〈85〉 bacterial endotoxins test - US Pharmacopeial Convention

Interim Revision Announcement
4 〈85〉 Bacterial Endotoxins Test Official April 1, 2011
Table 4. Preparation of Solutions for the Inhibition/Enhancement Test for Photometric Techniques
Solution to Which
Solution Endotoxin Concentration Endotoxin Is Added Number of Replicates
A a None Sample Solution Not less than 2
B b Middle concentration of the standard curve Sample Solution Not less than 2
C c At least 3 concentrations (lowest concentration is Water for BET Each not less than 2
designated λ)
D d None Water for BET Not less than 2
a Solution A: The Sample Solution may be diluted not to exceed MVD.
b Solution B: The preparation under test at the same dilution as Solution A, containing added endotoxin at a concentration equal to or near the middle of the
standard curve.
c Solution C: The standard endotoxin at the concentrations used in the validation of the method described for Assurance of Criteria for the Standard Curve
under Preparatory Testing (positive controls).
d Solution D: Water for BET (negative control).
PHOTOMETRIC QUANTITATIVE TECHNIQUES
Turbidimetric Technique
concentrations within the range indicated by the lysate
manufacturer to generate the standard curve. Perform the
assay using at least three replicates of each standard endotoxin
concentration according to the manufacturer’s instructions
for the lysate (volume ratios, incubation time, temperature,
pH, etc.). If the desired range is greater than two logs
This technique is a photometric assay measuring increases in the kinetic methods, additional standards should be inin
reactant turbidity. On the basis of the particular assay cluded to bracket each log increase in the range of the stanprinciple
employed, this technique may be classified as ei- dard curve. The absolute value of the correlation coeffither
an endpoint-turbidimetric assay or a kinetic-turbidimet- cient, r, must be greater than or equal to 0.980, for the
ric assay. The endpoint-turbidimetric assay is based on the range of endotoxin concentrations set up.
quantitative relationship between the concentration of endotoxins
and the turbidity (absorbance or transmission) of
the reaction mixture at the end of an incubation period.
The kinetic-turbidimetric assay is a method to measure either
the time (onset time) needed to reach a predetermined
absorbance or transmission of the reaction mixture, or the
rate of turbidity development. The test is carried out at the
incubation temperature recommended by the lysate manufacturer
(which is usually 37 ± 1°).
Test for Interfering Factors—Select an endotoxin con-
centration at or near the middle of the endotoxin standard
curve. Prepare Solutions A, B, C, and D as shown in Table 4.
Perform the test on Solutions A, B, C, and D at least in dupli-
cate according to the instructions for the lysate employed,
for example, concerning volume of Sample Solution and Ly-
sate TS, volume ratio of Sample Solution to Lysate TS, incu-
bation time, etc.
The test is considered valid when the following conditions
are met.
Chromogenic Technique
1. The absolute value of the correlation coefficient of the
standard curve generated using Solution C is greater than or
This technique is an assay to measure the chromophore
released from a suitable chromogenic peptide by the reaction
of endotoxins with lysate. On the basis of the particular
assay principle employed, this technique may be classified as
either an endpoint-chromogenic assay or a kinetic-chromogenic
assay. The endpoint-chromogenic assay is based on
the quantitative relationship between the concentration of
endotoxins and the release of chromophore at the end of
an incubation period. The kinetic-chromogenic assay is a
method to measure either the time (onset time) needed to
reach a predetermined absorbance of the reaction mixture,
or the rate of color development. The test is carried out at
the incubation temperature recommended by the lysate
manufacturer (which is usually 37 ± 1°).
equal to 0.980.
2. The result with Solution D does not exceed the limit of
the blank value required in the description of the lysate rea-
gent employed, or it is less than the endotoxin detection
limit of the lysate reagent employed.
Calculate the mean recovery of the added endotoxin by
subtracting the mean endotoxin concentration in the solu-
tion, if any (Solution A, Table 4), from that containing the
added endotoxin (Solution B, Table 4). In order to be consid-
ered free of factors that interfere with the assay under the
conditions of the test, the measured concentration of the
endotoxin added to the Sample Solution must be within
50% to 200% of the known added endotoxin concentration
after subtraction of any endotoxin detected in the solution
without added endotoxin.
When the endotoxin recovery is out of the specified
Preparatory Testing
range, the Sample Solution under test is considered to contain
interfering factors. Then, repeat the test using a greater
To assure the precision or validity of the turbidimetric and
chromogenic techniques, preparatory tests are conducted to
verify that the criteria for the standard curve are valid and
that the sample solution does not interfere with the test.
Validation for the test method is required when conditions
that are likely to influence the test result change.
Assurance of Criteria for the Standard Curve—The test
must be carried out for each lot of lysate reagent. Using the
Standard Endotoxin Solution, prepare at least three endotoxin
dilution, not exceeding the MVD. Furthermore, interference
of the Sample Solution or diluted Sample Solution not to ex-
ceed the MVD may be eliminated by suitable validated
treatment, such as filtration, neutralization, dialysis, or heat
treatment.To establish that the treatment chosen effectively
eliminates interference without loss of endotoxins, perform
the assay described above using the preparation to be examined
to which Standard Endotoxin has been added and
which has then been submitted to the chosen treatment.
© 2011 The United States Pharmacopeial Convention All Rights Reserved.